Genome-Wide Identification and Expression Analysis of the Trihelix Gene Family in Melon

doi:10.21203/rs.3.rs-4888918/v1

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Genome-Wide Identification and Expression Analysis of the Trihelix Gene Family in Melon

https://doi.org/10.21203/rs.3.rs-4888918/v1

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The trihelix transcription factor plays roles in regulating plant growth and development, and responses to various abiotic stresses. In this study, a comprehensive analysis of the trihelix family genes in Cucumis melo was conducted, identifying a total of 28 genes in its genome. These genes were further categorized into five subfamilies: GT-1, GT-2, SH4, GTγ, and SIP1. The study encompassed a systematic analysis of gene structures, chromosomal distribution, tandem and segmental duplication events, conserved motifs, phylogenetic relationships, and cis-acting elements of these trihelix genes. The syntenic analysis highlighted strong collinearity between trihelix family genes in C. melo with Arabidopsis, followed by rice while showing limited collinearity with those in B. distachyon. The cis-element analysis found 73 different types of cis-acting elements that are involved in stress tolerance, hormone signaling, growth and development, and responses to light in plants. Publicly available expression data of trihelix family genes in different tissues and growth stages were analyzed. The higher expressions of the genes, CmTH07, CmTH08, CmTH10, and CmTH14 in different tissues indicate their potential roles in growth and development. Based on cis-acting elements, and phylogeny study the genes CmTH02, CmTH16, CmTH20, CmTH21, CMTH24, CmTH26, and CmTH27 indicate their possible roles in abiotic stress tolerance. These findings contribute to enhancing our overall comprehension of the melon trihelix family. Furthermore, the research offers potential candidate genes to facilitate genetic enhancements for sustaining stress resistance in melons.

Bioinformatics

Cucumis melo

Genome-wide analysis

In-silico

Phylogeny

Trihelix

Transcription factors (TFs) are crucial in regulating plant growth, development, and responses to abiotic stresses (Riechmann et al. 2000). TFs regulate gene expression by selectively binding to promoter regions and interacting with cis-acting elements of genes (Lindemose et al. 2013; Mizoi et al. 2012). More than 60 TF families have been identified in plants (Jin et al. 2017; Wang et al. 2023). Trihelix is one of the first identified TF and is involved in diverse development processes and responses to abiotic stresses (Riechmann et al. 2000). It is also known as GT factors, and possesses a distinctive domain composed of three tandem helix structures (helix–loop–helix–loop–helix) that directly bind to GT elements essential for responses to light. This domain shares sequence similarity with MYB DNA-binding domains (Nagano 2000; Nagano et al. 2001; Zhou 1999). Based on the structure of the trihelical domain, trihelix TFs were classified into five distinct clades: GT-1, GT-2, SH4, GTγ, and SIP1 (Kaplan-Levy et al. 2012). Each α helix within the trihelix domain of GT-1 and SH4 features a tryptophan (W) residue in its internal hydrophobic region (Kaplan-Levy et al. 2012). While within the GT-2 and GTγ subfamilies, a consistent substitution occurs where a conserved tryptophan is replaced with phenylalanine (F), whereas in the SIP1 subfamily, this substitution is characterized by the replacement with isoleucine (I) (Kuhn et al. 1993; Xie et al. 2009).

The initially identified trihelix, TF GT-1 was isolated from the pea (Pisum sativum), and found to have a role in regulating light-dependent expression (Green et al. 1987). Homologous GT-1 family members were subsequently identified in Arabidopsis, rice, and tobacco. However, their physiological roles in the light response remained elusive (Ayadi et al. 2004; Gilmartin et al. 1992; Kaplan-Levy et al. 2012; Perisic and Lam 1992; Wang et al. 2004). In recent years, trihelix genes from diverse plants, including tomato, Arabidopsis, chrysanthemum, and rice, have been studied, revealing their involvement in various biological processes (Song et al. 2016; Yasmeen et al. 2016; Yu et al. 2015). The Arabidopsis PETAL LOSS (PTL) gene, a member of the GT-2 subfamily, plays a crucial role in controlling the growth of petals, sepals, and the overall morphogenesis of floral organs (Brewer et al. 2004; Griffith et al. 1999; Lampugnani et al. 2012). Arabidopsis ASIL1 functions as a temporal controller in seed filling (Gao et al. 2011), whereas Arabidopsis ASIL2 and rice LOC-Os02g3610 genes control the initial stages of embryo development. Rice seed shattering is regulated by a sole dominant trihelix gene known as SHATTERING1 (SHA1) (Lin et al. 2007). Populus PtaGTL1 plays a role in controlling both stomatal development and the absorption of water in plants (Yoo et al. 2019). The function of tomato SlGT11 involves the determination of floral organ types and the maintenance of floral traits (Yu et al. 2018).

Recent studies revealed that trihelix plays a crucial role in the diverse mechanisms that plants employ to cope with abiotic stresses. In Arabidopsis, the AtAST1 gene, belonging to the SIP1 subfamily, enhances resistance to salt and osmotic stresses (Xu et al. 2018). In addition, enhancing tolerance to abiotic stresses (e.g., salt, freezing, drought, etc.) is achievable by upregulating GmGT-2A and GmGT-2B gene expression (Xie et al. 2009). The transcription factor GTL1 in Arabidopsis controls both water use efficiency and drought tolerance (Yoo et al. 2010), whereas the GT-4 facilitates salt tolerance through its interaction with TEM2 (Wang et al. 2014). The OsGTγ-1 gene has been proven to exhibit a reaction to salt-induced stress in rice (Fang et al. 2010). ShCIGT plays a role in enhancing cold and drought tolerance in tomatoes through its interaction with SnRK1 (Yu et al. 2018). Overexpressing the BnSIP1-1 gene from the SIP1 clade in Brassica napus enhances seed germination when subjected to osmotic pressure, salt, and ABA treatments (Luo et al. 2017).

In contrast to other gene families in plants, the trihelix family is relatively small, typically consisting of 30–60 members in most species and around 100 members in individual polyploid species (Lang et al. 2024). For instance, there are 36 Trihelix genes in Solanum lycopersicum, 52 in Brassica rapa, 20 in Chrysanthemum morifoliym, 63 in Glycine max, and 94 in hexaploid wheat (Jin et al. 2014; Osorio et al. 2012; Song et al. 2016; Wang et al. 2017; Xiao et al. 2019; Yu et al. 2015).

C. melo L. is an annual diploid herb belonging to the Cucurbitaceae family (Napolitano et al. 2020). Melons have high nutritional value and significant economic importance (Amanullah et al. 2021; Amanullah et al. 2018). It is also well known for its medicinal properties, like antioxidant, anticancer, anti-inflammatory, analgesic, and diuretic effects, along with prevention activities of hypothyroidism, atherosclerosis, and other diseases (Gomez-Garcia et al. 2021; Lopes et al. 2020; Zhang et al. 2020). It is native to India and Africa (Yang et al. 2020). Melons are globally grown, generating a yearly harvest exceeding 27 million tons as of 2019 (Grumet et al. 2021). Melon farming faces a significant challenge as it requires a substantial amount of water for cultivation (Cabello et al. 2009). For optimal yield and fruit quality, it is advised to ensure that melon plants remain adequately hydrated without experiencing water stress from the flowering stage through the entire harvest period (Long et al. 2006). Saline soils originating from internal sources and the stress caused by salinity resulting from irrigation with saline water (Mangal et al. 2009) adversely affect the growth and production of melon. Though moderately tolerant to salty soils, melon yields vary under salt stress, influenced by the levels of stress, varieties of melon, and surrounding conditions etc. (Botía et al. 2005; Franco et al. 1997; Huang et al. 2012; Mangal et al. 2009; Tedeschi et al. 2011).

While the role of trihelix genes has been extensively explored in Arabidopsis, rice, and soybean, their specific roles in melon remain to be studied. The current study involved the comprehensive identification and in-silico characterization of the trihelix gene family in melon at a genome-wide level along with spatial and temporal expression analysis across various developmental stages, utilizing publicly available RNA-seq data in a way to identify the potential roles of trihelix genes in melon.

Retrieval of Trihelix genes from melon genomic databases

To obtain the candidate melon trihelix genes, four databases Melonomics v4.0 (https://www.melonomics.net/melonomics.html/) (Ruggieri et al. 2018), Cucurbit genomics CuGenDBv2 (http://cucurbitgenomics.org/v2/) (Yu et al. 2022) Plant transcription factor database (http://planttfdb.gaolab.org/index.php) (Jin et al. 2017), iTAK (http://itak.feilab.net/cgibin/itak/index.cgi) (Zheng et al. 2016) had been searched. According to the gene ID, identical genes were kept and repeated ones were deleted. The SMART (https://smart.embl.de/smart/batch.pl) (Schultz et al. 2000), NCBI CCD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.html) (Marchler-Bauer et al. 2015), and InterPro (https://www.ebi.ac.uk/interpro/) (Paysan-Lafosse et al. 2022) websites were used to confirm the trihelix domain in all the identified trihelix genes. All the sequences were downloaded from the CuGenDBv2 database (Supplementary table: ST2). The theoretical pI and Mw of the genes were analyzed by uploading protein sequences to ExPASy (https://web.expasy.org/compute_pi/) (Gasteiger et al. 2003). Cello v.2.5 (http://cello.life.nctu.edu.tw/) (Yu et al. 2014) was utilized to predict subcellular localization. The trihelix genes of Rice and Brachypodium distachyon, Arabidopsis were collected from the PlantTFDB website and the sequences were also downloaded from this site.

Sequences Alignment and Phylogenetic Analysis

MEGA11.0 (Tamura et al. 2021) was utilized to align the full-length sequences of trihelix proteins in melon, rice, Arabidopsis, and stiff brome. Alignment was done by ClusterW and the phylogenetic tree was constructed by the maximum likelihood (ML) method with a bootstrap value of 1000 replicates and default parameters iTOL (https://itol.embl.de/) (Letunic and Bork 2021) was used to edit it.

Multiple Sequence Alignments

Multiple sequence alignments of amino acid sequences of the trihelix genes of melon were perfectly constructed by COBALT (https://www.ncbi.nlm.nih.gov/tools/cobalt/re_cobalt.cgi) (Papadopoulos and Agarwala 2007) and it was visualized with BioEdit (Alzohairy 2011) (Supplementary File SF2).

Gene Position on Chromosomes and Gene Duplication Analysis

The information about CmTH genes, the position, and the length of chromosomes was obtained from the Melonomics database. TBtools (Chen et al. 2020) software was used to identify the position of the genes on chromosomes. The duplicate genes and the types of duplication were analyzed by the MCScanX tool and visualized with TBtools (Supplementary Table ST2).

Collinearity Analysis

To assess syntenic relations among trihelix family genes in melon, Arabidopsis, rice, and B. distachyon genomes, MCScanX with default parameters was employed, and visualized the results using TBtools software.

Conserved Structures, Motifs, and Cis-Acting Element Analysis

The exon and intron structure of trihelix genes were generated using the GSDS program (version 2.0) (http://gsds.gao-lab.org/) (Hu et al. 2015) with coding and genomic sequences. MEME suite software (version 5.5.5) (https://meme-suite.org/meme/) (Bailey et al. 2015) with the parameters of a maximum number of motifs of 10 was used to analyze the motifs of each deduced trihelix protein. The cis-acting elements of each CmTH gene up to 2000 bp upstream promoter region were predicted by using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al. 2002) and MS Excel 2019 was used to generate a heat map.

Expression Profile Analysis

MelonetDB (https://melonet-db.dna.affrc.go.jp/ap/mvw) (Yano et al. 2017) database was used to have the expressions of the identified genes. RNA-seq transcriptome studies data in 45 samples of the melon are available here. The heat map of the gene expression on the samples was conducted by the R programming via the “pheatmap” package with Euclidean distance and ward.D2 as the clustering model.

Comparative Modeling of Protein and Sequence Similarities

The process of comparative modeling, also known as homology modeling, was executed utilizing the Swiss model (https://swissmodel.expasy.org/interactive) (Schwede et al. 2003) platform through a systematic progression of four key phases: template selection, target-template alignment, model construction, and model assessment (Martí-Renom et al. 2000). Furthermore, a sequence similarity analysis was conducted using the TBtools, thereby enabling the comparison of amino acid sequences and the creation of a similarity matrix for comparative evaluation.

Interaction Network of CmTH Proteins

The web-based software String (https://string-db.org/ ) (Szklarczyk et al. 2023) was used to predict the protein-protein interaction of CmTH proteins. The k-means clustering method was set to visualize the interaction into three clusters.

The Trihelix gene in melon

A total of 108 sequences were retrieved from Melonomics, Cucurbit genomics, iTAK, and Plant Transcription Factor Database. These sequences of melon trihelix proteins were subjected to protein domain search in the NCBI CCD, SMART, and InterPro databases to confirm the presence of a complete trihelix domain. A total of 28 genes were identified to have a trihelix domain. Detailed information on these genes is listed in Table 1. The 28 trihelix genes were named CmTH01-CmTH28 according to the increasing trend of Molecular weights (MW) (He et al. 2004; Romano et al. 2004). The length of the open reading frames (ORFs) of trihelix proteins ranged from 110 aa to 927 aa, molecular weight from 12.91129 kDa to 102.71252 kDa, and the pI values from 4.81 to 9.77 (Table 1). The GRAVY value of these proteins ranged between − 0.36 to -1.252 (Table 1) which indicates that CmTH proteins are hydrophilic. The subcellular localization of these trihelix proteins in C. melo was predicted through the cello database. All these belong to the nucleus, except the CmTH28 has quite an equal probability of belonging to the nucleus or mitochondria (Table 1).

The PlantTFDB was utilized to identify trihelix genes within the genomes of A. thaliana, O. sativa, and B. distachyon for phylogenetic analysis. The initial numbers of identified genes were 34 in A. thaliana, 32 in O. sativa, and 54 in B. distachyon. After eliminating duplicate genes, 29 identical genes from A. thaliana, 30 from B. distachyon, and 32 in O. sativa, were selected for further analysis.

Conserved Structures, and Motifs

The conserved motifs of melon trihelix genes were analyzed by using the MEME Suite tool. A total number of 10 conserved motifs was identified (Supplementary file SF1). TBtools software was used to visualize the results (Fig. 1B). Motifs 2, 4, and 1 were found in almost all genes, and tryptophan (W) has been defined as an MYB/SANT-like DNA-binding domain because of its resemblance and overlap with the MYB transcription factor's helix-turned-angle helix structure (Yang et al. 2023). The GT-1 subfamily genes contained Motifs 2, 4, and 1. In the GT-2 subfamily motifs 10, 5, and 8 were located at the intermediate position and 1,3 motifs were located at the 3’ end. Also, other motifs were presented in this subfamily except motif 7. The GTɣ subfamily contained 2, 4, and 1 motifs at the intermediate position. In the SH4 subfamily, motif 2 was located at the 5’ end and motif 1 was located at the intermediate position and contained motif 8. The SIP1 subfamily included 2,4,1 genes located at the 5’ end with motif 7 located at the 3’ end. GSDS software was used to analyze the exon-intron structures of these genes (Fig. 1C). The exon number of melon trihelix genes varied from 1 to 18. In the GT-1 subfamily, the average number of exons was highest and the GTɣ subfamily had the lowest (only one) average number of exons each of the genes contained only one exon. The largest trihelix gene belonged to the GT-1 subfamily with the highest numbers of exons (18). GT-2 subfamily genes had 2–3 exons and only one had 5 exons. SH4 subfamily genes had 3–4 exons. Four genes in the SIP1 subfamily had only 1 exon, one had 6 exons and the other had 2–3 exons. A similar exon-intron pattern was observed among the members of the same subfamily with some exceptions.

Sequence alignment of trihelix

To identify the conserved ‘helix-loop-helix’ domain within trihelix proteins in melon, multiple sequence alignment was performed (Fig. 2). There is no evidence to show which sequence evolved first. All members of the five sub-families exhibited well-separated conserved domains for three helices. Two individual α-helices with conserved amino acid tryptophan (W) were identified in all the subfamilies, whereas in the third helix, the tryptophan was found replaced by phenylalanine (F) or isoleucine (I). The conserved amino acid cysteine (C) was also found in the third helix. MEME Suite analysis was used to predict three motifs within the trihelix domain. Each of them exhibited significant conservation (Fig. 3). The prediction indicated high conservation for two tryptophan residues in most of the sequences. These tryptophan residues play a role in creating helices, with one tryptophan present in each helix (Kaplan-Levy et al. 2012). Motif-2 also shows that Leucine (L) is another conserved residue (Fig. 3). In helix-2 serine (S) is conserved in all the subfamilies except SIP1, where alanine (A) is found as a conserved residue. Glutamine (Q) was conserved in all the subfamilies in helix-3 except the GT-2 subfamily.

Table 1

Basic Characteristics of *C. melo Trihelix* genes
Gene name	Gene ID	Chromosomal Location	Strand	Gene (bp)	CDS (bp)	Protein (aa)	Exon	Intron	Protein MW (Kda)	pI	GRAVY	Subcellular Localization
CmTH01	MELO3C028450	08 : 11367067–11368128	Forward	1062	333	110	5	4	12.91129	9.44	-0.471	Nucleus
CmTH02	MELO3C021031	01 : 24833494–24834751	Reverse	1258	837	278	1	0	32.35813	5.28	-1.064	Nucleus
CmTH03	MELO3C016254	07 : 21010443–21013071	Reverse	2629	840	279	2	1	33.22558	8.25	-1.117	Nucleus
CmTH04	MELO3C018722	01 : 2393682–2397716	Forward	4035	879	292	2	1	33.99259	9.64	-0.837	Nucleus
CmTH05	MELO3C035616	12 : 13742646–13746346	Reverse	3701	893	296	3	2	34.25723	9.63	-0.799	Nucleus
CmTH06	MELO3C009160	04 : 32646707–32648785	Forward	2079	918	305	2	1	35.98586	6.92	-1.115	Nucleus
CmTH07	MELO3C002654	12 : 20942204–20946065	Forward	3862	963	320	3	2	36.13677	5.99	-0.629	Nucleus
CmTH08	MELO3C022356	11 : 30650125–30652863	Reverse	2739	1068	355	3	2	38.74639	5.45	-0.692	Nucleus
CmTH09	MELO3C025995	08 : 28103645–28106265	Reverse	2621	972	323	2	1	38.83971	9.59	-1.037	Nucleus
CmTH10	MELO3C006287	06 : 2215453–2217022	Reverse	1570	1101	366	1	0	40.18232	9.33	-0.835	Nucleus
CmTH11	MELO3C003604	04 : 2493520–2496187	Forward	2668	1089	362	3	2	40.82397	5.16	-0.724	Nucleus
CmTH12	MELO3C024327	01 : 34872064–34873789	Forward	1726	1122	373	1	0	40.93779	9.67	-0.92	Nucleus
CmTH13	MELO3C012478	10 : 198804–201250	Reverse	2447	1152	383	2	1	42.15656	9.77	-0.961	Nucleus
CmTH14	MELO3C024229	01 : 34215449–34217496	Forward	2048	1158	385	1	0	44.60798	4.81	-1.244	Nucleus
CmTH15	MELO3C020079	10 : 11851125–11860097	Forward	8973	1185	394	4	3	44.65024	9.02	-0.966	Nucleus
CmTH16	MELO3C010724	03 : 28871494–28875296	Reverse	3803	1194	397	5	4	45.41276	6.05	-0.897	Nucleus
CmTH17	MELO3C014604	05 : 1025203–1028651	Forward	3449	1341	446	1	0	50.39453	6.36	-0.88	Nucleus
CmTH18	MELO3C012365	10 : 981355–985561	Forward	4207	1350	449	2	1	51.0013	7.19	-0.786	Nucleus
CmTH19	MELO3C008343	03 : 4073044–4075114	Reverse	2071	1350	449	1	0	51.4563	6.32	-1.043	Nucleus
CmTH20	MELO3C009422	04 : 31066914–31068491	Reverse	1578	1404	467	1	0	52.39846	5.92	-1.045	Nucleus
CmTH21	MELO3C023266	11 : 629359–631382	Forward	2024	1554	517	2	1	59.09312	5.91	-1.003	Nucleus
CmTH22	MELO3C009440	04 : 30926897–30930247	Forward	3351	1554	517	3	2	59.10911	6.85	-1.128	Nucleus
CmTH23	MELO3C009423	04 : 31060540–31065529	Reverse	4990	1617	538	7	6	59.25028	6.45	-0.557	Nucleus
CmTH24	MELO3C011683	03 : 10194329–10197119	Reverse	2791	1692	563	2	1	63.55282	5.92	-0.983	Nucleus
CmTH25	MELO3C005815	09 : 23225738–23228220	Reverse	2483	1815	604	2	1	67.72335	5.25	-0.916	Nucleus
CmTH26	MELO3C024673	02 : 20798333–20800981	Forward	2649	1965	654	2	1	75.90606	6.86	-1.252	Nucleus
CmTH27	MELO3C011694	03 : 9795534–9798858	Reverse	3325	2100	699	2	1	78.60632	6.32	-0.875	Nucleus
CmTH28	MELO3C026230	02 : 26459780–26470845	Reverse	11066	2784	927	18	17	102.71252	8.23	-0.36	Nucleus, Mitochondria

Gene Position on Chromosomes and Gene Duplication Analysis

Genome annotation files were used to identify the position of the genes on chromosomes. The 28 CmTH genes were distributed among 12 chromosomes of melon. Most of the chromosomes contained more than one gene. Chromosome 4 contained the highest number of genes (5) followed by Chromosome 1 and 3 (4 genes each). Chromosomes 2, 11, and 12 contained one pair of genes followed by chromosomes 5, 6, 7, and 9 having only one gene. The genes of the different subfamilies were mostly randomly distributed; for instance, 6 genes of the GT-1 subfamily were distributed on 6 different chromosomes (Fig. 3). Additionally, two pairs of segmental duplications were found on the melon genome (CmTH06/CmTH09 and CmTH04/CmTH07) (Fig. 3).

Cis-Acting Element Analysis

The promoter region cis-acting elements (CAEs) are associated with gene functions and expression patterns (Todeschini et al. 2014). Based on the function and response to stimuli all the predicted cis-acting elements were divided into four broad categories namely, light-responsive, phytohormone-responsive, abiotic and biotic stress-responsive, and plant growth and development-related. A total of 27 types of light-responsive elements (LREs) were revealed in the CmTH genes promoter region, among them Box-4, G-Box, G-box, GT1- motif, TCT-motif, and GATA-motif were prevalent (Fig. 4). CAEs were found in a total of 12 types, which revealed their association with hormone-specific expression. Abscisic acid-responsive element (ABRE) and ethylene-responsive element ERE were found with maximum abundance, with GARE- motif and AuxRR-core found with a lower percentage. Twenty types of CAEs were found in the abiotic and biotic stress-responsive category; among them ARE, MYB, MYC, and STRE were found in the highest percentage. The assessment of CmTH gene promoters revealed 12 CAEs associated with growth and developmental processes. Among them, AAGAA and as-1 motifs were found in maximum abundance while only the CmTH12 promoter region included the HD-ZIP 3 CAE, which is associated with cambium and vascular development.

Comparative Modeling of Protein and Sequence Similarities

In the field of computational biology, homology modeling utilizes the structural conservation observed within protein families to predict the three-dimensional atomic-resolution structure of a protein of interest by employing the experimentally determined structure of a homologous protein as a template. The process of protein structure homology modeling is predicated upon the evolutionary affinity shared between the target protein of interest and the template protein (Bordoli et al. 2009). The predicted structures of CmTH proteins, alongside their corresponding template data, are systematically organized (Supplementary File SF3). In protein homology modeling, the assessment metric known as GMQE (Global Model Quality Estimation) serves as a crucial measure for evaluating the credibility of derived models. The utmost value was identified in the instance of CmTH04, CmTH06, CmTH15, and CmTH28, whereas the minimal value was observed for CmTH01. The proteins within each subfamily exhibited congruent trends regarding the number of alpha helices and beta sheets (Supplementary File SF3). The comparative analysis of sequence homology among the CmTH proteins revealed a range from a minimum of 3.44 (CmTH01 compared to CmTH14) to a maximum of 51.99 (CmTH03 compared to CmTH06). Sequence similarity within subfamilies was also assessed, revealing that proteins belonging to similar subfamilies exhibit a higher percentage of similarity (Supplementary Table ST4).

Predicted protein-protein association networks of CmTH proteins

Protein-protein interaction was predicted to identify and categorize physical interactions between pairs or groups of trihelix proteins. All the proteins were categorized into three distinct clusters by their established associations. The analysis indicated that the core node-bearing proteins (like CmTH05) in the network may have potential participation in diverse functions through interacting with other proteins. The association networks showed that CmTH08 and CmTH28 may exhibit co-expression phenomena. Again, CmTH03 associated with CmTH05 and CmTH06 may show high protein homology (Fig. 5).

Phylogenetic analysis of trihelix genes

The PlantTFDB database was used to identify the trihelix genes in A. thaliana, O. sativa, and B. distachyon genomes to use in phylogeny analysis and a number of genes was 34, 32, and 54 respectively. After deleting the duplicate genes from A. thaliana and B. distachyon respectively 29 and 30 identical genes were used for further analysis. The relation of trihelix proteins in Arabidopsis, O. sativa, B. distachyon, and melon was shown as a maximum-likelihood phylogenetic tree using 119 trihelix sequences (Fig. 6). All the trihelix genes of melon were clustered into five major subfamilies namely SIP1, SH4, GTɣ, GT-2, and GT-1. The subgroups were named based on previous studies on Arabidopsis (Gao et al. 2009; Kaplan-Levy et al. 2012) and wheat (Xiao et al. 2019). The 28 CmTH genes were distributed in all the subgroups. Based on all four crop species SIP1 subfamily was the largest clade having 40 members and GTɣ subfamily was the smallest clade having 15 members. GT-2 subfamily contained the highest number (9 genes) of melon trihelix genes, followed by the SIP1 subfamily (8 genes). The GT-1 contained 5 genes. The lowest number of melon trihelix genes belonged to both SH4 and GTɣ subfamilies containing only 3 members each. In C. melo, two pairs of paralogous proteins were identified; CmTH21 and CmTH24; and CmTH06 and CmTH09. Additionally, the placement of the GT-1 and GT-2 at the bottom of the phylogenetic tree supports the hypothesis of diversification of these two subfamilies early in the existence of land plants (Qin et al. 2014).

Collinearity Analysis within CmTH Genes of Selected Species

To better comprehend the collinearity within the melon trihelix family (CmTH), we generated comparative syntenic diagrams involving melon and three other species (Arabidopsis, rice, and B. distachyon) (Fig. 7). The details of the collinearity are outlined in Supplementary Table ST2. The CmTH genes exhibited varying degrees of syntenic relationships with the three species, with the most pronounced collinearity observed with Arabidopsis followed by rice. Conversely, the collinearity relationship with B. distachyon was notably limited. Specifically, CmTH12 and CmTH05 genes demonstrated syntenic associations with Arabidopsis and rice, respectively. However, only two genes displayed collinearity with both rice and B. distachyon.

Expression profiling of trihelix genes in C. melo

The expression profile of the 28 trihelix genes in 45 tissues and conditions of melon encompassing various stages of roots, stems, leaves, flowers, regenerating calluses, ovaries, flesh, peel, and fruit development (Yano et al. 2017) were investigated using MelonetDB (Fig. 8, Supplementary Table ST3). The number of expressed genes showed limited variation, ranging from 18 to 22. The highest number of genes (22) were expressed in callus tissue, while the lowest number of genes (18) were expressed in the twelfth leaves, fruit flesh, and fruit epicarp. Some of the genes, including CmTH04, CmTH05, CmTH06, CmTH12, CmTH21, and CmTH22, showed no expression in any of the samples.

The gene expressions were categorized into three distinct clusters based on their expression level (Fig. 8). The first cluster includes 14 genes, consisting of both non-expressed and low-expressed genes. Certain genes exhibited higher tissue-specific expression like CmTH09 in fruit flesh (4 weeks after harvest), and CmTH15 in the shoot apex. Then 10 moderately expressed genes were found in the second cluster. Several genes from this cluster showed higher tissue-specific expression, including CmTH20 in callus, CmTH13 in fruit epicarp (4 weeks after harvest), and CmTH24 in the anther of female flower. The genes CmTH07, CmTH08, CmTH10, and CmTH14 showed higher levels of expression and were found in the last cluster. Among these CmTH08 and CmTH10 showed a higher level of expression in all fruit samples (Fig. 8)

Recent studies have indicated a substantial correlation between the trihelix gene and aspects such as growth, development, and tolerance to abiotic stresses in plants (Wang et al. 2016; Zhao et al. 2023a). Genome-wide exploration and expression analysis has been demonstrated in numerous studies to enhance the understanding of the origin, diversity, and biological function of particular gene families (Ohta 2000; Yang et al. 2023). This study provides a comprehensive genomic exploration of the genome-wide trihelix gene family in melon.

In the melon genome, a total of 28 genes were identified that have a complete trihelix protein domain. Gene duplication analysis of these genes suggested that melon trihelix genes exhibit a limited number of segmentally duplicated genes, specifically only two pairs, which account for 14.29% of the total 28 trihelix genes identified. Gene duplication serves as a prominent evolutionary mechanism, facilitating the emergence of new genes crucial for organisms to adapt to diverse environments (Kong et al. 2007; Moore and Purugganan 2003). The number of genes within TF families typically increases through the occurrence of tandem and segmental duplications (Kong et al. 2007). The finding of fewer segmental duplicated genes implies that the trihelix genes in melon likely have diverse origins and may not share a common ancestor. Other crops exhibited a comparable occurrence of segmentally duplicated genes including Medicago truncatula (2 pairs of segment duplication genes among 38 trihelix genes) (Liu et al. 2020). Similarly, among the 32 sesame trihelix genes, only 5 pairs of duplicate genes were found (Zhao et al. 2023b).

Trihelix genes were initially categorized into three distinct subfamilies: GTα, GTβ, and GTγ (Fang et al. 2010). Then, Kaplan-Levy et al. categorized trihelix genes in rice and Arabidopsis into five groups known as GT-1, GT-2, SH4, SIP1, and GTγ (Kaplan-Levy et al. 2012). In the current study, the melon trihelix genes were divided into five groups based on their evolutionary analysis with Arabidopsis. A similar grouping was also found in maize and M. truncatula (Liu et al. 2020; Zhao et al. 2023a).

Structural variations in genes are crucial for gene evolution, as the integration and rearrangement of gene fragments can lead to changes in the number of exons and introns (Xu et al. 2012). In this study, we detected a notable divergence in CmTH28 within the GT-1 subfamily, characterized by its 18 exons. In contrast, the majority of other genes in this subfamily are typically comprised of just two exons, with only one gene exhibiting a structure consisting of 5 exons. This outcome suggests that the particular CmTH28 gene has experienced a sequence of evolutionary events, resulting in an augmentation of exons, potentially indicating functional variations. Besides, the absence of introns in genes was identified within the SIP1 and GTγ subfamilies, a phenomenon previously noted in trihelix genes of Brassica napus, maize, and sorghum (Li et al. 2021; Zhang et al. 2022a; Zhao et al. 2023a). Consistent structures and conserved motifs were observed throughout the majority of genes within a specific subfamily, suggesting comparable functions and consistent evolutionary stability. However, the motif structures varied among distinct subfamilies. This result suggests that the five subfamilies may have different functions in melon.

The cis-acting elements in the promoter regions were analyzed to predict the functions of CmTH genes. At the transcription level, gene regulations are governed by the CAEs located in the promoter region (Koul et al. 2019). All the CmTH genes contained numerous light-responsive elements in their promoter regions, which is consistent with the reported light-responsive properties of the trihelix transcription factor family (Green et al. 1987; Kaplan-Levy et al. 2012; Zhou 1999). In Arabidopsis, the TCT motif is found to play the role of a positive photoperiod regulator by causing early flowering and a short growth period (Zhao et al. 2018). In plant development and abiotic/biotic stress response cascades, phytohormones mediate several essential biological processes (Shu et al. 2018). For anaerobic induction, the ARE motif is a crucial regulatory element. According to structural analysis, AREs (anaerobic responsive elements) are bipartite comprising GC and GT motifs. The GT motif resembles the low oxygen and dehydration-induced MYB2 transcription binding site (Dolferus et al. 2001). The MYB transcription factors have a major impact on abiotic stress tolerance, disease resistance, secondary metabolism, hormone signaling, and plant growth (Li et al. 2015). However, MYB genes are mostly involved in drought stress tolerance (Zhang et al. 2023). The jasmonic acid (JA) signaling pathway is regulated by MYC transcription factors, and is involved in the regulation of biotic and abiotic stresses, wounding response, plant growth and development, and secondary metabolite biosynthesis (Yanfang et al. 2018). Several CAEs have been found as drought-inducible cis-motifs implicated in the regulation of drought-responsive genes in plants, including STRE, DRE, LTR, and MBS (Niu et al. 2020). Growth-related CAEs, AAGAA, and as-1 were found as the most abundant. AAGAA plays a crucial role in xylem development and facilitates water transport whereas for numerous hormone-responsive genes, the as-1 element functions as a critical binding site, particularly in the context of auxin and methyl jasmonate stress signaling pathways (Maqsood et al. 2022).

Analyzing the pattern of gene expression in tissues is crucial for understanding the functional characteristics of genes. In this study, publicly available RNA-seq data were utilized to analyze the expression patterns of 28 melon trihelix genes. The majority of CmTH genes exhibited a non-tissue-specific expression in melon. The trihelix family plays important roles in various growth and developmental processes associated with flower (Li et al. 2008), stomata, trichrome, late embryogenesis, and also in the accumulation of glucan (Fan et al. 2018). The GT-2 subfamily of the trihelix gene is linked to floral organ morphogenesis (petal loss) in Arabidopsis (Li et al. 2008). It governs the development of petals and sepals, influencing their growth and the fusion of sepals (Brewer et al. 2004). In this research, the GT-2 subfamily CmTH12 displayed elevated expression levels in melon flowers, specifically in the anther and sepal regions. This heightened expression could potentially influence the development of floral organs in melons. Within the GT-2 subfamily, Arabidopsis AT1g33240 (GTL1) plays a role in the development of epidermal hairs, suggesting GT-2 subfamily genes may be involved in the formation of leaves, stems, and other organs (Jiang et al. 2020). CmTH27, orthologous of AT1g33240, is highly expressed in leaf samples in RNA expression analysis. This finding suggests that the CmTH27 gene may be strongly related to leaf formation. The elevated expression levels of CmTH08 and CmTH10 in the fruit suggest that they might play a pivotal role in the developmental processes of the fruit. Tissue-specific genes could be crucial for the development and specialization of specific organs or tissues, but further experiments are required to confirm the biological functions of these CmTH genes.

Due to the increasing frequency of climate change related extreme weather events, plants are encountering intensified challenges for survival, including situations of drought, salinization, and extreme temperature stresses (Zhang et al. 2022b). The trihelix TFs have been suggested to play a role in governing the regulatory processes associated with the tolerance to abiotic stresses especially against drought, salinity, water-logging, and low-temperature stresses (Du et al. 2015; Fang et al. 2010; Giuntoli et al. 2014; Luo et al. 2017; Magwanga et al. 2019; Xi et al. 2012). In Arabidopsis, AST1 (At3g24860) is crucial in mediating responses to salt and osmotic stresses (Xu et al. 2018). It will be interesting to see if its homologous gene in melon (CmTH02) does the same function. In addition, the DRE1 is present in CmTH02 indicating that CmTH02 may have a similar function in salt and osmotic stress tolerance as well. GT-2 (AtGTL1, At1g33240) suppresses SDD1, altering stomatal density and enhancing drought resistance, while GT-4 (AT3G25990) regulates Cor15A, contributing to salt tolerance (Wang et al. 2014). Genes CmTH27, CmTH21, and CmTH24 being homologous to At1g33240, and possessing MYB CAE indicates that these genes may show resistance against drought stress. Melon gene CmTH16, homologous to AT3G25990, may have contributed to salt tolerance. AtGT2 (At5G28300), the pioneering trihelix transcription factor binding to Ca2+/CaM−, plays a regulatory role in response to low temperature and salinity stress (Xi et al. 2012). It’s Orthologous gene CmTH26 may have a similar function against low temperature and salt stress in melon. HRA1 (At3G10040) identified as a submergence or hypoxia-responsive GTγ trihelix gene in Arabidopsis which functions as a suppressor of the ERF-VII transcription factor RAP2.12 that ultimately leads to the downregulation of key hypoxia-responsive genes (Giuntoli et al. 2014). In this current research, the identification of the CmTH20 gene within the GTγ family, exhibiting orthology to HRA1 having maximum ARE, implies its potential involvement in conferring flood tolerance.

The melon genome revealed the presence of 28 members of the trihelix gene family which were categorized into 5 subfamilies through phylogenetic analysis. Similar gene structures and conserved functional domains were observed among genes within the same subfamily with some exceptions. Two pairs of segmentally duplicated genes were identified. Expression profiles of the trihelix genes during growth, development, and exposure to environmental stresses such as flooding, high-salt conditions, low temperature, and drought indicated the potential candidate genes conferring tolerance to these stresses. The findings of this study may serve as a basis for future research aiming at unravelling the exact functions of CmTH genes in melon. These genes, given their potential roles is abiotic stress tolerance, may be targeted for developing stress-tolerant varieties in the future.

Ethical approval

Not applicable.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Funding

This study was supported by the Bangladesh Bureau of Educational Information & Statistics (BANBEIS), Ministry of Education, Bangladesh (Project No: 2022/24/MoE) and the Bangladesh Agricultural University, Mymensingh-2202 (Grant No: 2024/2117/BAU). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Author Contributions

MRH was responsible for conceptualization and fund acquisition. MNAS and MJHJ were responsible for data collection and curation. MNAS was responsible for analyzing and writing the original draft. MKB contributed to synteny analysis. MRH was responsible for fund acquisition, supervising the work, reviewing and finalizing the draft. All authors have read and agreed to the final version of the manuscript.

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The authors declare no competing interests.

SF1MotifLogo.pdf
SF1 - Motif Logo
SF2SequenceAlignment.pdf
SF2 - Sequence Alignment
SF3ProreinStructure.pdf
SF3-Prorein Structure
ST1MelonTrihelixSequences.xlsx
ST1 - Melon Trihelix Sequences
ST2GeneDuplicationandColinearity.xlsx
ST2 - Gene Duplication and Collinearity
ST3RNAExpression.xlsx
ST3 - RNA Expression
ST4SequenceSimilarities.xlsx
ST4- Sequence Similarities

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Genome-Wide Identification and Expression Analysis of the Trihelix Gene Family in Melon

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Retrieval of Trihelix genes from melon genomic databases

Sequences Alignment and Phylogenetic Analysis

Multiple Sequence Alignments

Gene Position on Chromosomes and Gene Duplication Analysis

Collinearity Analysis

Conserved Structures, Motifs, and Cis-Acting Element Analysis

Expression Profile Analysis

Comparative Modeling of Protein and Sequence Similarities

RESULTS

Conserved Structures, and Motifs

Sequence alignment of trihelix

Gene Position on Chromosomes and Gene Duplication Analysis

Cis-Acting Element Analysis

Comparative Modeling of Protein and Sequence Similarities

Predicted protein-protein association networks of CmTH proteins

Phylogenetic analysis of trihelix genes

Collinearity Analysis within CmTH Genes of Selected Species

DISCUSSION

CONCLUSION

Declarations

Ethical approval

Funding

Author Contributions

References

Additional Declarations

Supplementary Files

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